Quercetin attenuates cisplatin-induced ovarian toxicity in rats: Emphasis on anti-oxidant, anti-inflammatory and anti-apoptotic activities

2.1 Drugs and chemicals

Quercetin (purity: 98%) was obtained from Merck (St. Louis MO, USA). Cisplatin was obtained as Cisplatine® vials for injection (1 mg/1 mL, Mylan, Italy). Further chemicals were of the highest available grade.

2.2 Animals and study design

Thirty female Wistar rats (200–225 g) were obtained from our animal facility, King Abdulaziz University (KAU). Rats were housed on a 12-h light–dark cycle at 23 ± 2 °C. Animal hanling was approved Faculty of Pharmacy’s Research Ethics Committee, King Abdulaziz University (# PH-1442-63).

Rats were randomly divided into five groups (6 each) and treated for seventeen sequential days as follows: Group 1 (Control): animals were treated once daily with the vehicle (5% DMSO in corn oil; 5 mL/kg p.o.); Group 2 (Quercetin; Q): rats were treated once daily with the quercetin (dissolved in 5% DMSO in corn oil) at a dose of 10 mg/kg using the oral gave route; Group 3 (Cisplatin; CP): rats were treated once daily with the vehicle (5% DMSO in corn oil; 5 mL/kg p.o.). Besides, cisplatin group was administered a single intraperitoneal (i.p.) injection on days 7 and 14 with cisplatin (6 mg/kg).; Group 4 (CP + Q 5 mg/kg) and group 5 (CP + Q 10 mg/kg): rats were treated once daily with the quercetin (dissolved in 5% DMSO in corn oil) at a dose of 5 mg/kg or 10 mg/kg respectively. Furthermore, cisplatin group was given a single i.p. injection on days 7 and 14 with cisplatin (6 mg/kg). The doses of cisplatin and quercetin were chosen based on a pilot experiment and in accordance with previous studies (Qin et al., 2019; Said et al., 2019).

At 24 h after the last injection, rats were anesthetized by an i.p. injection of ketamine at a dose of 100 mg/kg and xylazine (10 mg/kg). Blood samples were collected from the retro-orbital sinus and allowed to coagulate. Sera were then separated by centrifugation at 3500 rpm for 15 min and stored at −80 °C for subsequent analyses. After blood collection, the rats were euthanized by decapitation. Then, abdomens were sterilized with povidone-iodine solution and opened longitudinally. Ovarian tissues were quickly dissected out, and washed with ice-cold PBS. Part of the ovaries were kept in 10% neutral-buffered formalin for subsequent histological and immunohistolochemical studies. The rest of the ovarian tissues were homogenized in PBS (0.1 M, pH 7.4), then centrifuged at 10,000 rpm at 4 °C for 15 min. Subsequently, supernatants were collected and stored at −80 °C.

2.3 Histopathological examination

Ovaries were kept in 10% formalin/saline for 24 h, then paraffin blocks were prepared by standard procedure. Then, they were sectioned (5 µm thickness). And stained using hematoxylin and eosin (H&E). Slides were examined and photographed using a light microscope (Nikon Eclipse TE2000-U, NIKON, Japan). Examinations were performed by a pathologist unaware of the treatment protocol. Follicles were distinguished as primordial, pre-antral, antral, and atretic as described previously (Britt et al., 2000; Said et al., 2019). Healthy follicles (%) was determined based on the following: $$\mathrm{P}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e}\mathrm{h}\mathrm{e}\mathrm{a}\mathrm{l}\mathrm{t}\mathrm{h}\mathrm{y}\mathrm{f}\mathrm{o}\mathrm{l}\mathrm{l}\mathrm{i}\mathrm{c}\mathrm{l}\mathrm{e}\mathrm{s}=\frac{\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{p}\mathrm{r}\mathrm{i}\mathrm{m}\mathrm{o}\mathrm{r}\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{l}+\mathrm{p}\mathrm{r}\mathrm{e}-\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{l}+\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{l}\mathrm{f}\mathrm{o}\mathrm{l}\mathrm{l}\mathrm{i}\mathrm{c}\mathrm{l}\mathrm{e}\mathrm{s}}{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{f}\mathrm{o}\mathrm{l}\mathrm{l}\mathrm{i}\mathrm{c}\mathrm{l}\mathrm{e}\mathrm{s}}\times 100$$

2.4 Biochemical analysis

Serum estradiol, anti-Müllerian hormone (AMH) and active caspase-3 were assessed using MyBiosource ELISA kits (Cat No. MBS263466, MBS9712690 and MBS7244630) (MyBiosource, San Diego, CA, USA) according to the manufacturer instructions. The ELISA analytical biochemical technique of the used kits is based on respective antibody-antigen interactions (immunosorbency) and a horseradish peroxidase (HRP) colorimetric detection system to detect the respective antigen targets in samples. Malondialdehyde (MDA) and reduced glutathione (GSH) contents, superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzyme activities, and total protein were determined using commercially available kits (Biodiagnostic, Cairo, Egypt). Tumor necrosis factor-α (TNF-α) was estimated using rat ELISA kits (Cat. No. E-EL-R0019) obtained from Ellabscience, (Houston, TX, USA). The method depends on adding samples to the micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for rat TNF-α and Avidin-HRP conjugate were added successively to each micro plate well and incubated. The substrate was added to each well. Biotinylated antibody and avidin-HRP conjugate appeared blue in color. The optical density (OD) was measured spectrophotometrically at a wavelength of 450 nm and compared to a standard curve.

2.5 Immunohistochemical analyses

Following standard procedures for tissue preparations, slides were incubated overnight at 4 °C with the primary antibodies: rabbit polyclonal anti-NFκB (p65) (catalog # ab16502), anti-COX-2 (catalog # ab15191), and anti-IL-6 (catalog # ab271269) (ABCAM, Cambridge, UK). Slides were then flushed and incubated with the secondary anti-rabbit antibody (catalog # CTS005, R&D systems, MN, USA) (Kim et al., 2016). Images were analyzed as optical density (OP) using appropriate software (Image J, 1.46a, NIH, USA) using at least 3 sections per rat. Details of the protocol used in our laboratory have been previously described (Algandaby et al., 2017).

2.6 Real-time polymerase chain reaction (RT-qPCR) analysis of Bax and Bcl-2

Ovarian tissues were homogenized and RNA was extracted using a commercial kit and quantified. Reverse transcription was achieved using A cDNA Reverse Transcription Kit (catalog # 4368814, Applied Biosystems, Foster City, CA, USA). PCR was performed using Taq PCR Master Mix Kit (catalog # 201443, Qiagen, Valencia, CA, USA). Bax, and Bcl2 expression was determined in relation to β-actin. Sequence of forward and reverse primers of is illustrated in Table 1. After the RT-PCR run, the data were given in the cycle threshold (Ct). The relative quantitation (RQ) of each gene to β-actin was determined based on determining delta-delta Ct (ΔΔCt) (Livak and Schmittgen, 2001).

2.7 Statistical analysis

Data are presented as mean (M) ± SD. One-way ANOVA followed by Tukey’s test were used for comparing group means. Significance was taken at p < 0.05. GraphPad Prism version 8 (GraphPad, La Jolla, CA, USA) was used for statistical tests.

3.1 Effect of quercetin against cisplatin-induced histological ovarian injury

Microscopic examination of ovarian tissue from the normal group (Fig. 1A) revealed normal histologic structure; the ovarian cortex contained different stages of developing follicles including primordial, primary, secondary and graafian follicles and the ovarian stroma was composed of collagenous fibers. In resemblance to the normal group, administration of quercetin (Fig. 1B) showed normal ovarian tissue. In cisplatin group (Fig. 1C), ovaries tissue showed numerous luteal structures occupying the ovarian cortex, intense inflammatory edema in the ovarian stroma with decreased numbers of developing follicles. This was evidenced by decreased proportion of primordial, pre-antral, and antral follicles and increased fraction of atretic follicles. However, daily administration of quercetin (5 mg/kg) exhibited an ameliorative action against cisplatin-induced ovarian toxicity, the normal developing follicles occupied most of the ovarian cortex with few luteal structures despite the intense inflammatory reaction observed in most of the examined cases (Fig. 1D). Co-administration of quercetin (10 mg/kg) achieved higher activities in protecting the ovarian tissue against cisplatin insult with lower inflammatory processes and higher proportion of healthy follicles (Fig. 1E). Semi-quantitative histological evaluation of follicles in the different groups indicated that cisplatin significantly impaired follicular maturation and resulted in almost 45% reduction of healthy follicles. However, quercetin (5 and 10 mg/kg) enhanced the proportion of healthy follicles by 40% and 61% in comparison to the cisplatin group (Fig. 1F).

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Fig. 1

Histopathological effects of quercetin on cisplatin-induced ovarian injury in rats. A: Control, showing different types of ovarian follicles; B: Control + Quercetin (Q), showing luteal structure (black arrow) and graafian follicle (black arrowhead); C: Cisplatin (CP), showing the luteal structures (blue stars) and the intense interstitial inflammatory reaction (red arrows); D: CP + Q (5 mg/kg), showing different stages of developing follicles, with scattered inflammatory cells infiltration; Hematoxylin &Eosin (H&E). E: CP + Q (10 mg/kg), showing normal graafian follicle with mild inflammatory cells infiltration; F: Semi-quantitative evaluation of % healthy follicles. Data are presented as Mean ± SD (n = 6). ^a Significant difference from Control group at p < 0.05. ^b Significant difference from CP at p < 0.05. ^c Significant difference from CP + Q (5 mg/kg) at p < 0.05.

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3.2 Effect of quercetin on cisplatin-induced hormonal alterations

Serum estradiol levels were not significantly altered in the different study groups (Fig. 2A). To assess the impact of cisplatin on the number of follicular pool in the ovaries, serum levels of AMH was determined. Cisplatin significantly lowered AMH values by 45% compared to control group. However, treatment with quercetin significantly ameliorated the decline of AMH levels by 36% and 81% compared to the cisplatin group.

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Fig. 2

Effects of quercetin on serum levels of AMH (A) and estradiol (B) in cisplatin-treated rats. Data are presented as Mean ± SD (n = 6). ^a Significant difference from Control group at p < 0.05. ^b Significant difference from CP at p < 0.05. ^c Significant difference from CP + Q (5 mg/kg) at p < 0.05.

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3.3 Effect of quercetin on oxidative status

The data in Table 2 indicate that ovaries from animals in the cisplatin group showed enhanced MDA content by 68%, depletion of GSH by 38% and exhaustion of SOD and GPx activities by 43% and 36%, respectively as compared to control values. Ovarian tissues collected from animals treated with quercetin (5 mg/kg) showed inhibition of MDA accumulation by 25% and enhancement of GSH content by 63% as well as elevation of SOD and GPX activities by 38 and 40% when compared to cisplatin group. Quercetin in the higher dose (10 mg/kg) almost normalized GSH content and SOD and GPx activities.

3.4 Assessment of NFκb, Cox-2 and IL-6 expression immunohistochemically

The data in Fig. 3A indicate that ovarian tissues from cisplatin-treated animals showed relatively higher expression of NFκb. However, treatment with quercetin (5 and 10 mg/kg) ameliorated the rise in NFκb expression by 10% and 42% respectively. Also, expression of the inflammation markers COX-2 and IL-6 were significantly enhanced by cisplatin. The higher dose of quercetin (10 mg/kg) significantly inhibited COX-2 and IL-6 expression by 48% and 38% respectively (Fig. 3B&C).

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Fig. 3

Effect of quercetin on expression of NFκb, COX-2 and IL-6 in ovarian tissues of cisplatin-treated rats. Data are presented as Mean ± SD (n = 6). ^a Significant difference from Control group at p < 0.05. ^b Significant difference from Q at p < 0.05. ^c Significant difference from CP at p < 0.05. ^d Significant difference from CP + Q (5 mg/kg) at p < 0.05.

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3.5 Effect of quercetin on TNF-α in ovaries of cisplatin-treated rats

As shown in Fig. 4, quercetin alone did not significantly alter TNF-α contents as compared to control group. However, cisplatin insult resulted in a significant increase of ovarian tissues by 140% as compared to control values. Nevertheless, quercetin at doses of 5 and 10 mg/kg significantly prevented the rise in TNF-α content by 16% and 42% respectively, as compared to cisplatin group.

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Fig. 4

Effect of quercetin on expression of TNF-α content in ovarian tissues of cisplatin-treated rats. Data are presented as Mean ± SD (n = 6). ^a Significant difference from Control group at p < 0.05. ^b Significant difference from Q at p < 0.05. ^c Significant difference from CP at p < 0.05. ^d Significant difference from CP + Q (5 mg/kg) at p < 0.05.

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3.6 Impact of quercetin on cisplatin-induced ovarian apoptosis and follicle loss

Cisplatin significantly enhanced apoptotic processes in ovarian tissues as it resulted in approximately 2-fold increase in cleaved caspase-3 content. However, quercetin treatment at doses of 5 and 10 mg/kg inhibited caspase-3 increase by 22% and 42% respectively, as compared to cisplatin group (Fig. 5A). This was confirmed by assessing mRNA expression of Bax and Bcl2. Cisplatin significantly enhanced Bax expression by 105% and inhibited Bcl2 expression by 53% as compared to control animals. The higher dose of quercetin (10 mg/kg) significantly prevented the rise in Bax mRNA expression by 38% and the inhibition of Bcl2 expression by 75%, as compared to cisplatin group (Fig. 5B&C). The protective effects of quercetin were further confirmed by the significant elevations in the Bax/Bcl2 ratio observed in treatment groups as compared to cisplatin-alone group (Fig. 5D).

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Fig. 5

Effect of quercetin on expression of caspase-3 content and mRNA expression of Bax and Bcl2 in ovarian tissues of cisplatin-treated rats. Data are presented as Mean ± SD (n = 6). Statistical analysis was performed by one-way ANOVA followed by Tukey test. ^a Significant difference from Control group at p < 0.05. ^b Significant difference from Q at p < 0.05. ^c Significant difference from CP at p < 0.05. ^d Significant difference from CP + Q (5 mg/kg) at p < 0.05.

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